The present invention relates to a composition comprising a plurality of cDNAs which are differentially expressed in Alzheimer""s disease and which may be used entirely or in part to diagnose, to stage, to treat, or to monitor the treatment of a subject with a brain disorder.
Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for examining which genes are tissue specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder.
The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with Alzheimer""s disease may be compared with the levels and sequences expressed in normal brain tissue.
Alzheimer""s disease is a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibrillary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain. The hippocampus is part of the limbic system and plays an important role in learning and memory. In subjects with Alzheimer""s disease, accumulating plaques damage the neuronal architecture in limbic areas and eventually cripple the memory process.
Approximately twenty million people worldwide suffer with dementia that results from Alzheimer""s disease. The disease can be early onset affecting individuals as young as 30 years of age, or it can be familial or sporadic. Familial Alzheimer""s disease was once thought to be inherited strictly as an autosomal dominant trait; however, this view is changing as more genetic determinants are isolated. For example, some normal allelic variants of apolipoprotein E (ApoE), which is found in senile plaques, can either protect against or increase the risk of developing the disease (Strittmatter et al. (1993) Proc Natl Acad Sci 90:1977-1981).
Mutations in four genes are known to predispose an individual to Alzheimer""s disease: ApoE, amyloid precursor protein (APP), presenilin-1, and presenilin-2 (Selkoe (1999) Nature 399:A23-A31). The e4 allele of the ApoE gene confers increased risk for late onset Alzheimer""s disease. xcex2-amyloid protein (Axcex2) is the major component of senile plaques, and it is normally formed when xcex2- and xcex3-secretases cleave APP. In Alzheimer""s disease patients, large quantities of Axcex2 are generated and accumulate extracellularly in these neuropathological plaques. Efforts to understand the mechanism underlying Axcex2 deposition have recently focused on the APP-cleaving secretases. In fact, two yeast aspartyl proteases have been shown to process APP in vitro (Zhang et al. (1997) Biochim Biophys Acta 1359:110-122). Evidence using peptidomimetic probes further confirms that the secretases are intramembrane-cleaving aspartyl proteases (Wolfe et al. (1999) Biochemistry 38:4720-4727). The presenilin-1 gene is a candidate for the xcex3-secretase that cleaves the APP carboxyl terminus. Several lines of evidence support the involvement of presenilins in the disease process. Presenilin can be coimmunoprecipitated with APP, and mutations in the presenilin genes increase production of the 42-amino acid peptide form of Axcex2. These missense point mutations result in a particularly aggressive, early onset form of the disease (Haas and DeStrooper (1999) Science 286:916-919).
The proteases, BACE1 and BACE2 (xcex2-site APP cleaving enzymes 1 and 2) which appear to be xcex2-secretases, are potential therapeutic targets because of their ability to cleave APP. Vasser et al. (1999; Science 286:735-741) have found that BACE1 is an aspartyl protease with xcex2-secretase activity which cleaves APP to produce Axcex2 peptide in vitro. It is expressed at moderate levels across all brain regions and is concentrated in neurons but not in glia. BACE2, which has 52% amino acid identity with BACE1, has been described by Saunders et al. (1999; Science 286:1255a). Whereas BACE1 maps to the long arm of chromosome 11, BACE2 maps to the Down syndrome region of chromosome 21 (Acquati et al. (2000) 468: 59-64; Saunders et al. supra). This location is significant because middle-aged Down syndrome patients have enhanced xcex2-amyloid deposits. Other members of the BACE family may also participate in this APP cleavage: the amino terminals of Axcex2 peptides appear to be cleaved heterogeneously indicating that there may be several xcex2-secretases involved in APP processing (Vasser (1999) Science 286:735-741).
Associations between Alzheimer""s disease and many other genes and proteins have been reported. Fetal Alzheimer antigen (FALZ) and synuclein a (SNCA) are found in brain plaques and tangles. Inheritance of some gene polymorphisms is also linked to increased risk of developing the disease. For example, a polymorphism in the gene encoding xcex22-macroglobulin, a protein that can act as a protease inhibitor, is associated with increased risk for developing a late-onset form of Alzheimer""s disease.
The present invention provides for a composition comprising a plurality of cDNAs for use in detecting changes in expression of genes encoding proteins that are associated with Alzheimer""s disease. Such a composition can be employed for the diagnosis, prognosis or treatment of Alzheimer""s disease and possibly other forms of dementia correlated with differential gene expression. Differential gene expression may also reflect inflammation, proliferation, and glial cell activation which occur secondary to the disease process. The present invention satisfies a need in the art in that it provides a set of differentially expressed genes which may be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of a subject with a brain disorder such as Alzheimer""s disease.
The present invention provides a composition comprising a plurality of cDNAs and their complements which are differentially expressed in brain tissues and which are selected from SEQ ID NOs:1-138 as presented in the Sequence Listing. In one embodiment, each cDNA is downregulated at least two-fold, SEQ ID NOs: 1-95; in another embodiment, each cDNA is upregulated at least two-fold, SEQ ID NOs:96-138. In one aspect, the composition is useful to diagnose a brain disorder selected from akathesia, Alzheimer""s disease, amnesia, amyotrophic lateral sclerosis, ataxias, bipolar disorder, catatonia, cerebral palsy, cerebrovascular disease Creutzfeldt-Jakob disease, dementia, depression, Down""s syndrome, tardive dyskinesia, dystonias, epilepsy, Huntington""s disease, multiple sclerosis, muscular dystrophy, neuralgias, rneurofibromatosis, neuropathies, Parkinson""s disease, Pick""s disease, retinitis pigmentosa, schizophrenia, seasonal affective disorder, senile dementia, stroke, Tourette""s syndrome and cancers including adenocarcinomas, melanomas, and teratocarcinomas, particularly of the brain. In another aspect, the composition is immobilized on a substrate.
The invention also provides a high throughput method to detect differential expression of one or more of the cDNAs of the composition. The method comprises hybridizing the substrate comprising the composition with the nucleic acids of a sample, thereby forming one or more hybridization complexes, detecting the hybridization complexes, and comparing the hybridization complexes with those of a standard, wherein differences in the size and signal intensity of each hybridization complex indicates differential expression of nucleic acids in the sample. In one aspect, the sample is from a subject with a brain disorder and differential expression determines an early, mid, and late stage of that disorder.
The invention further provides a high throughput method of screening a library of molecules or compounds to identify a ligand. The method comprises combining the substrate comprising the composition with a library of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand. Libraries of molecules or compounds are selected from DNA molecules, RNA molecules, mimetics, peptides, transcription factors and other regulatory proteins.
The invention still further provides an isolated cDNA selected from SEQ ID NOs:16, 34, 59, 86, and 126 as presented in the Sequence Listing. The invention also provides an expression vector comprising the cDNA, a host cell comprising the expression vector, and a method for producing a protein comprising culturing the host cell under conditions for the expression of a protein and recovering the protein from the host cell culture. The invention additionally provides a method for purifying a ligand, the method comprising combining a cDNA of the invention with a sample under conditions which allow specific binding, recovering the bound cDNA, and separating the cDNA from the ligand, thereby obtaining purified ligand.
The present invention provides a purified protein encoded and produced by a cDNA of the invention. The invention also provides a high-throughput method for using a protein to screen a library of molecules or compounds to identify a ligand. The method comprises combining the protein or a portion thereof with the library of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. Libraries of molecules or compounds are selected from DNA molecules, RNA molecules, PNAs, mimetics, peptides, proteins, agonists, antagonists, antibodies or their fragments, immunoglobulins, inhibitors, drug compounds, and pharmaceutical agents. The invention further provides for using a protein to purify a ligand. The method comprises combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and separating the protein from the ligand, thereby obtaining purified ligand. The invention still further provides a pharmaceutical composition comprising the protein. The invention yet still further provides a method for using the protein to produce an antibody. The method comprises immunizing an animal with the protein or an antigenically-effective portion thereof under conditions to elicit an antibody response, isolating animal antibodies, and screening the isolated antibodies with the protein to identify an antibody which specifically binds the protein.
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The Sequence Listing is a compilation of cDNAs obtained by sequencing and extension of clone inserts. Each sequence is identified by a sequence identification number (SEQ ID NO) and by the clone number (Clone ID) from which it was obtained.
Table 1 lists the cDNAs which are differentially expressed, downregulated at least two-fold, by their SEQ ID NO, Accession number, Clone ID, and by the description associated with at least a fragment of a cDNA found in GenBank. The descriptions were obtained using the sequences of the Sequence Listing and BLAST analysis.
Table 2 lists the cDNAs which are differentially expressed, upregulated at least two-fold, by their SEQ ID NO, Accession number, Clone ID, and by the description associated with at least a fragment of a cDNA found in GenBank. The descriptions were obtained using the sequences of the Sequence Listing and BLAST analysis.
Table 3 lists the source of the RNAs used to produce cDNAs for hybridization to the UNIGEM V microarray (Incyte Pharmaceuticals, Palo Alto Calif.). The columns present the Tissue, Gender, and medical conditions as known for each donor, Person 1 and Person 2.